From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells
Winter, E.M.
Citation
Winter, E. M. (2009, October 15). From cardiogenesis to cardiac regeneration : focus on epicardium-derived cells. Retrieved from https://hdl.handle.net/1887/14054
Version: Corrected Publisher’s Version
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
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Grauss RW1, Winter EM2, van Tuyn J1,3, Pijnappels DA1, Vicente Steijn R1, Hogers B2, van der Geest RJ4, de Vries AAF3, Steendijk P1, van der Laarse A1, Gittenberger-de Groot AC2, Schalij MJ1, Atsma DE1
Mesenchymal stem cells from ischemic heart disease patients improve left ventricular function after acute myocardial infarction
American Journal of Physiology. 2007; 293(4): H2438-H2447
6
Abstract
BackgroundMesenchymal stem cells (MSCs) from healthy donors improve cardiac function in experimental acute myocardial infarction (AMI) models. However, little is known about the therapeutic capacity of human MSCs (hMSCs) from patients with ischemic heart disease (IHD). Therefore, the behavior of hMSCs from IHD patients in an immune-compromised mouse AMI model was studied.Methods
Enhanced green fluorescent protein (eGFP)-labeled hMSCs from IHD patients (hMSC group: 2 × 105 cells in 20 μl, n=12) or vehicle only (Medium group: n=14) were injected into infarcted myocardium of NOD/scid mice. Sham-operated mice were used as the control (n=10). Cardiac anatomy and function were serially assessed using 9.4-T magnetic resonance imaging (MRI); 2 weeks after cell transplantation, immunohistological analysis was performed.
Results
At day 2, delayed-enhancement MRI showed no difference in myocardial infarction (MI) size between the hMSC and Medium groups (33±2% versus 36±2%; p: not significant). A comparable increase in left ventricular (LV) volume and decrease in ejection fraction (EF) was observed in both MI groups. However, at day 14, EF was higher in the hMSC than in the Medium group (24±3%
versus 16±2%; p<0.05). This was accompanied by increased vascularity and reduced thinning of the infarct scar. Engrafted hMSCs (4.1±0.3% of injected cells) expressed von Willebrand factor (16.9±2.7%) but no stringent cardiac or smooth muscle markers.
Conclusions
hMSCs from patients with IHD engraft in infarcted mouse myocardium and preserve LV function 2 weeks after AMI, potentially through an enhancement of scar vascularity and a reduction of wall thinning.
Introduction
Cell therapy is a promising treatment modality for patients with ischemic heart disease (IHD).
Recent, clinical studies showed a beneficial effect on left ventricular (LV) function after application of unselected bone marrow-derived mononuclear cells (BM-MNC) in the setting of acute myocardial infarction (AMI) 1 or in patients with chronic myocardial ischemia 2.
BM-MNCs are a heterogeneous group of cells containing >99.9% committed or differentiated cells, as well as a small fraction of uncommitted cells including mesenchymal stem cells (MSCs) 3. After being injected into ischemic myocardium, bone marrow (BM)-derived MSCs from various animal species can differentiate into multiple cell lineages, including endothelial cells 4 and cardiomyocytes
5,6, thereby improving LV function 4-6. In contrast, less information is available about the potential therapeutic capabilities of MSCs from human origin (hMSCs). In rat AMI models, transplantation of hMSCs from healthy subjects across xenogeneic barriers with or without immune suppression improved LV function 7,8. Also, in the only clinical post-MI trial described to date, Chen et al found that intracoronary infusion of autologous BM-derived MSCs resulted in an improved LV function 9. However, little is known about engraftment, differentiation potential and therapeutic capacity of hMSCs from IHD patients after transplantation in an ischemic myocardial environment.
Previous studies demonstrated that BM-MNC and endothelial progenitor cells (EPC) from IHD patients exhibit reduced migratory and colony-forming activities in vitro and resulting in less neovascularization in an ischemic hind limb mouse model compared with cells from healthy individuals 10. Also, risk factors for IHD correlate with reduced numbers and functional activity of circulating EPCs 11. At present, it is unclear if the presence of numerous cardiovascular risk factors in IHD patients affects the therapeutic potential of their hMSCs. As a diminished regenerative capability of hMSCs from IHD patients potentially limits their therapeutic efficacy, knowledge on the behaviour of these cells in ischemic myocardium is of interest.
The availability of the immunodeficient non-obese diabetic severe combined immunodeficient (NOD/
scid) mouse strain allows the investigation of hMSCs in animals without pharmacological immune suppression which might interfere with the injected cells 12. Furthermore, in recent years magnetic resonance imaging (MRI) techniques have been developed, allowing accurate and reproducible determination of murine cardiac volume and infarct size 13. The high spatial and temporal resolution combined with the non-invasive nature of MRI offer new insights about the dynamic changes induced by cardiac stem cell therapy by serial assessment of cardiac morphology and function.
In the present study, we investigated whether hMSCs from patients with IHD are able to i) engraft and survive in the acutely infarcted myocardium of NOD/scid mice, ii) prevent remodeling and improve LV function of the infarcted hearts, and iii) differentiate into endothelial cells, smooth muscle cells (SMCs) or cardiomyocytes after myocardial engraftment.
Methods
Animal experiments
Experiments were performed in 8 to 10 weeks old male immunodeficient NOD/scid mice (Charles River Laboratories, Maastricht, the Netherlands), which lack the ability to mount an adaptive B- and T-cell mediated immune response. All experiments were approved by the Committee on Animal Welfare of the Leiden University Medical Center (LUMC), the Netherlands. Animal experiments were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996).
BM harvest, hMSC isolation, expansion and labeling
hMSCs were purified from leftover BM samples of 4 adult IHD patients with drug-refractory angina and myocardial ischemia who were enrolled in ongoing clinical stem cell trials 2 as previously described 14. Briefly, BM was aspirated from the posterior iliac crest after local anesthesia, after which the mononuclear cell fraction (BM-MNC) was isolated by Ficoll density gradient centrifugation.
Twenty-four hours after seeding of the BM-MNCs in culture flasks, non-adherent cells were removed and adherent hMSCs were expanded by serial passage. A FACS flow cytometer and CellQuest software (Becton Dickinson, Franklin Lakes, NJ, USA ) were used to characterize the hMSC surface antigen profile, as previously described 15. hMSCs abundantly expressed hyaluronate receptor (CD44), major T-cell antigen (Thy-1;CD90), endoglin (CD105), vascular cell adhesion molecule-1 (CD106),and human leukocyte class I (HLA-ABC) antigens. These cells also expressed low levels of transferrin receptor (CD71), P-selectin (CD62P), ß3 integrin (CD61), neuralcell adhesion molecule (CD56), and membrane cofactor proteinof the complement system (CD46) at their surface. Furthermore, hMSCs showed 100
% differentiation into adipocytes and osteoblasts after appropriate stimulation, confirming their mesenchymal and multipotent nature (data not shown).
To facilitate their identification in vivo, hMSCs of passages 4 to 6 were transduced with 100 infectious units (IU) per cell of a fiber-modified first-generation human adenovirus serotype 5 vector (hAd5/F50.
CMV.eGFP) encoding the enhanced green fluorescent protein (eGFP), as previously described 15 in the presence of 5 mM sodium butyrate to enhance transgene expression.
Surgical protocol
Animals were preanesthetized with 5% isoflurane in a gas mixture of oxygen and nitrogen. After endotracheal intubation and ventilation (rate 200 breaths/min, stroke volume of 200 +l, Harvard Apparatus), a left anterior thoracotomy was performed, and the left anterior descending coronary artery (LAD) was ligated. After 15 min, 20 μl culture medium (M199, Eurobio, Cedex, France) containing 2 × 105 hMSCs (MI + hMSC group; n=12) or no cells (MI + Medium group; n=14) were injected at 5 sites in the infarcted area and border zones using a 20-μl syringe with a 33-gauge needle (Hamilton Company, Reno, NV). To determine baselines, 10 animals were prepared in a similar manner but without tightening the suture around the LAD (sham group).
MRI
Cardiac anatomy and function were serially assessed 2 and 14 days after AMI using a small animal MRI (Bruker BioSpin, Rheinstetten, Germany). The system consisted of a vertical 9.4-T (400 MHz), 89-mm bore nuclear magnetic resonance spectrometer equipped with a shielded gradient set (1 T/m). A birdcage radiofrequency coil with an inner diameter of 30 mm (Bruker BioSpin) was used to transmit and receive the nuclear magnetic resonance signals. Before imaging, mice were anesthetised as described above. Mice were then placed supine in a coil with a pneumatic pillow for respiration monitoring and maintained at 1 to 2% isofluorane. Electrocardiogram (ECG) electrodes were attached to the left fore limb and right hind limb. Biotrig software (Bruker BioSpin) was used to acquire ECGs and to measure respiratory rates. First, scout images for long axis orientation of the heart were obtained. Images containing a four-chamber view were next used to plan the short axis images.
Image reconstruction was performed using ParaVision 3.02 software (Bruker BioSpin).
Contrast-enhanced imaging
To determine myocardial infarct size at day 2 post-MI, contrast-enhanced MRI imaging was employed.
To this end, 150 μL (0.05 mmol/ml) gadolinium-diethylenetriamine-pentaacetic acid (Gd-DPTA, Dotarem) was injected via the tail vein. A high-resolution ECG- and respiratory-triggered two- dimensional fast-gradient echo (FLASH) sequence was used to acquire a set of 18 contiguous 0.5 mm slices in the short axis orientation covering the entire heart. Imaging parameters were: echo time (TE)
of 1.9 ms, repetition time (TR) of 90.5 ms, 25.6 mm x 25.6 mm field of view, matrix size of 256 × 256 and a flip angle of 60°.
LV Function
At day 2 and 14 after MI, LV function was assessed. A FLASH cine sequence was used to acquire a set of contiguous 1 mm slices in short axis orientation covering the entire long axis of the heart. Imaging parameters were the same as listed above, except for the TR, which was 7 ms, and the flip angle, which was 15°.
Image analysis
MRI images were analyzed with the MR analytical software system (MASS) for mice (MEDIS, Leiden, the Netherlands). The endocardial and epicardial borders were traced manually by 2 independent investigators who were blinded from the experimental groups. End-diastolic and end-systolic phases and the contrast-enhanced area were identified automatically, after which the percentage of infarcted myocardial volume, LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV) and LV ejection fraction (LVEF) were computed.
Histological examination
At day 15 after MI, the mice were weighed and euthanized, and their hearts and lungs were removed.
After measuring their wet weight, the lungs of each animal were freeze-dried. The difference between the wet and dry weight of each pair of lungs was used as a measure of pulmonary congestion. The hearts were immersion-fixed in 4% paraformaldehyde and embedded in paraffin. Serial transverse sections of 5 μm were cut across the entire long axis of the heart and subsequently mounted on slides. hMSC engraftment was assessed using antibodies (Abs) against eGFP (A11122, Invitrogen), and vascular endothelial cells were detected by platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31)-specific Abs (clone MEC13.3, Pharmingen), followed by appropriate secondary biotinylated Abs.
For visualization of the eGFP-specific Abs, we employed the ABC staining kit (Vector Laboratories) and the PECAM-1-specific signal was amplified using the CSA system (Dako). 3,3’-Diaminobenzidine tetrahydrochloride (DAB, Sigma-Aldrich) was used as substrate for horseradish peroxidase. Sections were counterstained with Mayer’s hematoxylin. The number of engrafted hMSCs was determined by counting the DAB-positive cells with a 20x magnification in every 10 th serial section of the entire long axis of the heart. The number of counted cells was multiplied by 10 to obtain an estimate of the total number of engrafted cells in the heart. The hMSC engraftment rate was subsequently calculated by dividing this number by the number of injected hMSCs (2 × 10 5 cells) and multiplying the result by 100%.
Assessment of cell differentiation
To investigate differentiation of eGFP-labeled hMSCs toward endothelial cells, SMCs or
cardiomyocytes, serial sections were incubated with Abs against human-specific von Willebrand factor (vWF; 4400-5884, Biogenesis), α-smooth muscle actin (ASMA; clone 1A4, A2547, Sigma-Aldrich), human specific smooth muscle myosin heavy chain (smMHC; clone HSM-V, M7786, Sigma-Aldrich), α-sarcomeric actin (clone 5C5, A2172, Sigma-Aldrich), sarcomeric myosin heavy chain (MHC; clone MF20, Hybridoma Bank, Iowa City, IA) and cardiac troponin I (cTnI; clone 19C7, 4T21, HyTest). Primary Abs were visualized with appropriate secondary biotinylated Abs followed by Qdot 655 streptavidin- conjugated (Q10121MP, Invitrogen) Abs. The eGFP-specific labeling was detected with an antibody against eGFP (A11122, Invitrogen) followed by an Alexa Fluor 488 antiserum. Colocalization of eGFP and differentiation markers was examined using a Nikon eclipse E800 fluorescence microscope (NIKON Europe, Badhoevedorp, The Netherlands) equipped with dedicated Q-dot compatible filter sets.
In vitro hMSC culture
To compare the protein expression of in vitro cultured and in vivo injected hMSCs, a small fraction of eGFP-transfected hMSCs from the same cell batch that was injected in the mice was propagated ex vivo. At the start and termination of the in vivo experiment, the cells maintained in vitro were analyzed by immunofluorescence staining 15 using the same aforementioned antibodies and the Nikon eclipse E800 fluorescence microscope.
Measurement of vascular density
The effect of hMSC transplantation on vascular density was determined by quantifying the number of PECAM-1-positive vessels per squared millimeter in both the infarcted border zone and infarcted scar area. Measurements were performed on 3 equidistant sections between the apex and ligature (at the midpoint between the LAD ligature and the apex, between the midpoint and the LAD ligature, and between the midpoint and the apex) from 5 animals per group. Per section, the number of PECAM-1-positive vessels in 8 equally distributed areas of 0.1 mm2 in the infarcted anterolateral wall of the LV and 6 equally distributed areas of 0.1 mm2 in both border zone areas were counted in a blinded fashion at a 20x magnification. The values were then expressed as number of vessels per mm2. All measurements were performed by two independent examiners who were blinded from the experimental groups using the Image-Pro Plus software package (Media Cybernetics, Carlsbad, CA).
Assessment of infarcted wall thickness
To measure thickness of the infarcted wall in both infarcted groups, a planimetric analysis of the same sections used for assessment of vascular density was performed using a drawing microscope (Olympus BH-2, Olympus America Inc.). Wall thickness was measured at 2 separate border zone areas and at the midpoint of the infarct region and averaged for all 3 measurements. Measurements were performed perpendicular to the infarcted wall.
Statistical analysis
Numerical values were expressed as means±SEM. Comparisons between the sham, MI+Medium and MI+hMSC groups were made using one-way analysis of variance (ANOVA), followed by unpaired t-tests between groups. A p value <0.05 was considered significant.
Results
Myocardial infarct size as assessed by MRI
Two days after LAD ligation, all mice in the MI+Medium (n=14) and MI+hMSC (n=12) groups underwent contrast–enhanced MRI with Gd-DPTA (Figure 1A) to determine the extent of the MI. Infarct size did not differ between the 2 groups (Figure 1B, 36±2% vs. 33±2% of the LV; p: not significant [ns]), indicating that hMSC transplantation had no acute effect on infarct size.
Cardiac function as assessed by MRI
LV function was characterized by serial cine MRI 2 and 14 days after LAD ligation in both MI groups and time-matched sham-operated controls. Representative examples of systolic and diastolic short axis views after sham operation, as well as 2 and 14 days after MI are shown in Figure 2. Two days after MI, a comparable increase in LVEDV (62±2 μL and 58±2 μL; p: ns) and LVESV (43±2 μL and 40±3 μL; p: ns) was measured as well as considerable decrease in LVEF (31±2% and 33±3%; p: ns) in both the MI+Medium and MI+hMSC groups. All values were significantly different from the sham group (LVEDV: 48±2 μL, LVESV: 24±1 μL and LVEF: 51±1%; p<0.05; Figure 3). At 14 days post-MI, LVEDV (130±8 μL versus 115±12 μL; p: ns; Figure 3, top) and LVESV (90±12 μL versus 110±8 μL; p: ns, Figure 3, middle) increased further in both the MI+Medium and MI+hMSC groups, although a non-significant trend toward an attenuated LVESV increase was observed in the MI+hMSC group. Of interest, at day 14, a significant difference was found between the LVEF in the MI+hMSC group (24±3%) and the LVEF in the MI+Medium group (16±2%; p<0.05; Figure 3, bottom).
Figure 1. Infarct size as assessed by delayed-enhancement MRI. (A) Gd-DPTA-enhanced MR image 2 days after myocardial infarction (MI, left panel), after tracing the endocardial and epicardial borders (middle panel), and after automatic quantification of infarct size by the MASS for mice software package (right panel). (B) No difference in infarct size was found 2 days after MI between the Medium and hMSC group. ns: not significant.
Figure 2. Typical transverse short axis MR images of the NOD/scid mouse heart at end-diastole (ED, A-C) and end-systole (ES, D-F) in sham-operated mice (A and D) and in mice 2 (B and E) and 14 (C and F) days after myocardial infarction (MI).
Figure 3. Anatomical and functional analysis of hearts from sham-operated animals (Sham), animals receiving medium only (MI + Medium), and animals receiving hMSCs (MI + hMSC) 2 days and 14 days after myocardial infarction (MI), as assessed by high-resolution 9.4-T MRI. Top: left ventricular end-diastolic volume. Middle: left ventricular end-systolic volume. Bottom: left ventricular ejection fraction. Data are expressed as mean±SEM. *: p<0.05 versus time-matched medium-treated mice, †: p<0.05 versus time-matched sham-operated animals.
Body and lung weight
Body weight before to the operation was similar in all 3 groups (sham group: 26.5±0.5 g, MI+Medium group: 26.8.±0.5 g, and MI+hMSC group: 25.8±0.6 g). Two weeks after MI, body weight in the MI+Medium group decreased significantly with 3.4±1.0 g (-12±4%) as compared to the sham group, which gained body weight with 0.3±0.4 g (1±1%; p=0.007; Figure 4A). In contrast, the body weight loss of 0.8±1.0 g (-3±4%; p: ns) in the MI+hMSC group was comparable to the sham group. The loss in body weight in the MI+Medium group was accompanied by an increase in lung fluid (p<0.05), which was not observed in the MI+hMSC group (Figure 4B).
Figure 4. Change in body weight (A) and amount of pulmonary fluid (B) at 2 weeks after induction of myocardial infarction (MI).
There is a significant decrease in body weight 2 weeks after MI in the MI+Medium group but not in the MI+hMSC group (A). The amount of lung fluid was increased in the medium-treated group but not in the mice that received hMSCs (B).*: p<0.05 versus sham-operated animals.
Figure 5. Immunohistochemical staining of eGFP-labeled hMSCs from ischemic heart disease (IHD patients) in the anterolateral wall 2 weeks after intramyocardial injection shows substantial engraftment of injected cells.
hMSC engraftment and differentiation
Two weeks after transplantation, an engraftment rate of 4.1±0.3% (n=5) of eGFP-labeled hMSCs was identified in hearts of the hMSC-treated animals (Figure 5). They were detected predominantly in the infarcted anterolateral wall and border zone of the infarcted area. No hMSCs were present in the non-infarcted posterior and septal walls. Serial sections were assessed to identify eGFP-positive cells co-expressing differentiation markers. The infarcted myocardium contained hMSCs positive for the human specific endothelial cell-specific protein vWF (16.9±2.7%) and the SMC marker ASMA (78.3±4.0%) (Figure 6). The engrafted hMSCs did not stain positive for the highly specific human SMC marker smMHC or the cardiomyocyte-specific proteins α-sarcomeric actin, cTnI and MHC (Figure 6).
No eGFP-labeled cells were found incorporated into the blood vessels.
In vitro differentiation of hMSCs
Before injection, hMSCs already stained positive for ASMA, but not for vWF, smMHC, cTnI or MHC (data not shown). Two weeks after in vitro culture, the cells were still positive for ASMA (Figure 7), but not for any other marker described above. This finding indicates that the ischemic in vivo environment may be responsible for the expression of vWF genes in the injected transplanted hMSCs.
Vascular density and wall thickness
Vascular density in the infarcted scar area and border zone areas was compared between the two MI groups 14 days after cell administration. Vessel diameter ranged from 8 to 94 μm in all groups. The total blood vessel density (as determined by the number of PECAM-1-positive vessels per mm2) in the scar area was significantly higher in the MI+hMSC group (610±78/mm2, n=5) than in the MI+Medium group (347±56/mm2, n=5; p<0.05; Figure 8A-C). Furthermore, also in border zone areas of the infarcted hearts, vessel density was increased in the MI+hMSC group (810±68/ mm2) compared with the MI+Medium group (565±50/mm2, p<0.05; Figure 8C). In addition, measurements of wall thickness showed that hMSC injection significantly reduced the extent of infarct wall thinning (MI+Medium group: 18.0±2.1 × 10-2 mm, MI+hMSC group: 30.5.0±2.9 × 10-2 mm; p<0.05; Figure 8A, B and D).
Figure 6. Assessment of differentiation of intramyocardially injected hMSCs from patients with ischemic heart disease (IHD) toward endothelial cells, smooth muscle cells or cardiomyocytes using immunofluorescence microscopy. The left column shows engrafted hMSCs in green (Alexa 488) and nuclei in blue (Hoechst), the middle column shows staining of the indicated protein expression in red (Qdot 655), and the right column is a merge of both images. Yellow arrows indicate host vascular media stained by α-smooth muscle actin (ASMA). vWF: von Willebrand factor, smMHC: smooth muscle myosin heavy chain, MHC: myosin heavy
Figure 7. hMSCs from the batches that were used for the transplantations exhibit α-smooth muscle actin (ASMA, top) but not von Willebrand factor (vWF, bottom) staining after in vitro culture for the duration of the animal experiment. Before transplantation, hMSCs already expressed the ASMA gene (data not shown). Nuclei are stained blue; ASMA and vWF are stained red.
Figure 8. Representative photographs of platelet endothelial cell adhesion molecule-1 (PECAM-1) staining of the infarct scar in animals treated 2 weeks earlier with medium only (A) or with hMSCs from ischemic heart disease (IHD) patients (panel B). Infarct scar and border zone vascularity was significantly increased in the hMSC group compared with the Medium group (C), and was associated with a reduction in infarct scar wall thinning at 2 weeks post-MI (D). *: p<0.05 versus time-matched medium-treated mice.
Discussion
Key findings of the present study are that in an immune-compromised mouse model of acute MI, intramyocardial injection of hMSCs from patients with IHD resulted in i) a significant preservation of LVEF compared with medium-treated animals, ii) no limitation of the early infarct size iii) an increased vascularity of the infarct scar iv) a marked reduction in the thinning of the infarcted wall, and v) differentiation of hMSCs toward endothelial cells but not toward cardiomyocytes or SMCs. The present data therefore demonstrate the feasibility of IHD-patient derived hMSCs in cell-based therapy for acute myocardial infarction.
Although a beneficial effect of autologous MSC transplantation in different animal models of IHD was demonstrated in several studies 4-6,16, little information is available about the therapeutic potential of hMSCs from patients with IHD. This is of particular interest because recent studies demonstrated that when compared with healthy controls, human BM-MNCs from patients with IHD have a reduced neovascularization capacity 10, and that risk factors for coronary artery disease correlate with reduced numbers and functionality of circulating hEPCs 11. In other words, limited functionality of cells from IHD patients may limit the potential use of these cells in the treatment of patients with AMI.
LV function and anatomy after hMSC transplantation
In the present study, cardiac function and morphology were assessed with a high-resolution 9.4T MRI scanner. MRI is a non-invasive technique that uses intrinsic contrast and, unlike one-dimensional (M-mode) and two-dimensional echocardiography, is capable of obtaining true three-dimensional anatomical and function information. Combined with the high temporal resolution, which enables accurate assessment of cardiac function, MRI is the imaging modality of choice to study the effects of stem cell therapy 17. In addition, the established clinical MRI technique of infarct size determination by delayed-contrast enhancement imaging after Gd-DPTA was recently adapted and validated in the mouse MI model 13. In the present study, no difference in infarct size between the hMSC and Medium groups 2 days after MI was found. This was consistent with the functional data showing at 2 days after MI no difference in any of the functional parameters between the MI+Medium and MI+hMSC groups. These results are in line with the recent findings that after transplantation of hMSCs from healthy volunteers in acutely infarcted rat hearts, hMSCs had no effect on LV function and remodeling measured 3 days after transplantation by two-dimensional echocardiography 7. However, at 2 weeks after MI, a significant preservation of LVEF in the MI+hMSC group as compared to the MI+Medium group was observed. Furthermore, we found a non-significant trend toward less LVESV increase in the MI+hMSC group compared with the non-treated group.
The deterioration in cardiac function in the MI+Medium group was associated with a significant increase in lung fluid, which is indicative of pulmonary congestion. In contrast, after hMSC treatment the amount of lung fluid was similar to that in sham-operated animals. Furthermore, the Medium group showed a significant loss of weight of more than 12% in contrast to only 3% in the hMSC group. Cardiac cachexia is defined as a weight loss of more than 7.5% of the original body weight and is a sign of end-stage heart failure 18. Therefore, these findings further substantiate the beneficial effects of hMSC injection from IHD patients on the preservation of LV function after AMI.
Phenotypical characterization of transplanted hMSCs
VWF-positive donor cells were detected after injection of hMSCs in infarcted mouse hearts, whereas in vitro cultured hMSCs from the same batch that was used for the in vivo experiments were negative for vWF at 2 weeks post-AMI. Therefore, it is likely that hMSCs acquired this endothelial cell marker in the ischemic myocardial environment. This finding is consistent with a previous study from Zhang et al, in which healthy human donor cells stained positive for vWF at 60 days but not at 3 days after injection of hMSCs in the acutely infarcted myocardium of immune-suppressed rats 7. In addition,
several studies reported the differentiation of animal MSCs into endothelial cells after acute and chronic myocardial ischemia 4,6.
In this study, hMSCs also stained positive for ASMA, which is commonly used as a marker for SMCs. ASMA-positive donor cells were detected after transplantation of MSCs in the ischemic rat
19 and canine 4 heart. It was striking, however, that in our study the hMSCs were already positive for ASMA prior to their injection and kept expressing the ASMA gene in vitro for at least the duration of the animal experiments. In agreement with our findings, Cai et al showed through immunohistochemistry and Western blot analysis that lapine and canine MSCs in monolayer cultures had ASMA incorporated into stress fibbers 20. Furthermore, reverse transcription-polymerase chain reaction data showed that in vitro cultured hMSCs already contain ASMA-specific transcripts 15. ASMA gene expression may thus be considered to be intrinsic to these cells. At 2 weeks after transplantation the hMSCs did not stain positive for smMHC, which is a highly specific marker for SMCs 21. We hence conclude that transplanted hMSCs did not acquire a true SMC phenotype. It cannot be excluded that the injected hMSCs have acquired characteristics of myofibroblasts, which have also been described to be ASMA positive but negative for smooth muscle myosin 22.
In the present study, none of the engrafted hMSCs were positive for the cardiac proteins cTnI, α-sarcomeric actin, or MHC. This is consistent with a previous study in dogs where 4 weeks after MI, none of the engrafted canine MSCs expressed the muscle-specific gene encoding desmin or the cardiac marker gene cardiac troponin T (cTnT) 4. Interestingly, Dai et al recently demonstrated in a chronic rat myocardial infarction model that allogeneic MSCs did not express muscle-specific marker genes two weeks after injection but did stain positive for the (striated) muscle markers α-actinin, MHC, phospholamban, and tropomyosin after 6 months 6. In contrast, Mangi et al demonstrated that autologous MSCs expressed MHC, cTnI, α-sarcomeric actin, and myosin light chain 3 weeks after acute MI in a rat model 23. Furthermore, other studies also demonstrated cardiomyogenic differentiation of porcine 5 and human 7 MSCs within 2 weeks after transplantation. The MSCs in these latter studies were obtained from healthy subjects. It is not clear whether the failure of injected hMSCs to differentiate into cardiomyocytes in our experiments is due to the fact that these cells were obtained from IHD patients, and this warrants further investigation.
Possible mechanisms of preservation of LV function by hMSCs from IHD patients
Since no transdifferentiation of hMSCs into cardiomyocytes was observed, it is likely that hMSCs exert their cardioprotective effects via other mechanisms than cardiomyocyte regeneration.
Interestingly, in a recent study of Nakamura et al injection of long-term cultured porcine MSCs into the acutely infarcted NOD/scid mouse heart resulted in functional improvement at 2 weeks despite minimal differentiation into cardiomyocytes (5 0.2% of the engrafted cells expressed cTnT) 24. The authors found an increased capillarity in the peri-infarct area and hypothesized that a possible trophic mechanism must be the basis of the observed beneficial effects 24.
In mice treated with hMSCs from IHD patients, we observed that vessel density in the infarcted scar and border zone was significantly higher than in medium-treated animals. Although we found some evidence for the endothelial differentiation of hMSCs, none of these cells were incorporated into vascular structures in the scar area. This indicates that in our study the endothelial covering of the vessels in the scar is derived from host tissue. In contrast, in other studies transplanted MSCs that expressed endothelial markers were found in the vessel lining 4,19. The reason for this discrepancy, however, is unclear. Of note, since vascular density was assessed 14 days after MI in the present study, it could be confounded by an inflammatory response to the transplanted hMSCs. However, the use of the immune-compromised NOD/scid mouse model in the present study makes this less likely.
Furthermore, Zhang et al demonstrated in a rat model of acute MI that an increase in vascularization
could be observed as early as 7 days after transplantation of hMSCs from healty volunteers, in contrast to the transplantation of human fibroblasts 25.
The transplanted MSCs may also have preserved LV function by secreting cytokines acting in a paracrine fashion. Recently, it was shown that rat MSCs, engrafted in ischemic myocardium, secrete angiogenic factors including stromal cell-derived factor-1α (SDF-1α), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor (bFGF), which may explain the increase in capillary density 16. The MSC-mediated increase in vascular structures may increase blood flow within the infarcted area and border zone and thus contribute to i) salvage of the ischemic myocardium, ii) inhibition of cardiac remodeling, iii) a gradual recruitment of hibernating cardiomyocytes, and iv) a subsequent improvement in systolic function. From human studies it is known that significant changes in LVEF can be observed as early as 10 days after revascularization of hibernating myocardium
26. The observed attenuation of infarct wall thinning in the hMSC group may result from inhibition of cardiac remodeling due to the improved myocardial perfusion. In turn, this may lead to a decrease in wall stress and, subsequently, decreased O2 consumption 27. This phenomenon may also account for the improved contractile performance of the hMSC-treated hearts. The increased vascularity observed in this study is in line with the improved myocardial perfusion found in clinical studies with BM-MNC 2.
Limitations
One of the limitations of the present study is the lack of a control group with hMSCs derived from stringently matched healthy subjects, which limits conclusions about a possible beneficial or detrimental effect of the presence of IHD per se on hMSCs therapeutic potential. However, it should be noted that even among healthy volunteers, considerable variation in stem cell characteristics may exist, potentially hampering an accurate delineation of IHD-induced alterations in stem cell function from normal biological variation 28. Furthermore, the aim of our study was to determine the feasibility of using hMSCs from IHD patients. This research does not aim, however, to perform a detailed comparison of these cells to hMSCs derived from healthy individuals. Nevertheless, future studies should be performed to assess the potential differences in therapeutic potential between hMSCs from IHD patients and healthy individuals. Another limitation of this study is the use of a model of acute MI with permanent ligation of the LAD, which does not reflect contemporary medical practice where patients with an MI undergo early reperfusion of the culprit artery. However, this reproducible model has been well established in literature and allows comparison with previously reported data. Furthermore, only the short-term effects (2 weeks) of hMSC transplantation were studied.
We used MRI to assess anatomy and function, which resulted in a rather high mortality because of long acquisition times under general anesthesia. This was especially the case in the animals with severe heart failure, making longer-time follow-up with the current protocol practically impossible.
Nevertheless, long term studies are warranted.
Conclusions
hMSCs from patients with IHD engraft in infarcted mouse myocardium and preserve LV function 2 weeks after acute myocardial infarction, potentially through enhancement of scar vascularity and reduction of wall thinning. hMSCs were found to express endothelial cell markers, but no stringent cardiac or smooth muscle markers.
Reference List
1.
Schachinger V, Erbs S, Elsasser A, Haber- bosch W, Hambrecht R, Holschermann H, Yu J, Corti R, Mathey DG, Hamm CW, Suselbeck T, Assmus B, Tonn T, Dimmeler S, Zeiher AM. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med. 2006; 355:1210-1221.
2.
Beeres SL, Bax JJ, Dibbets-Schneider P, Stokkel MP, Fibbe WE, van der Wall EE, Schalij MJ, Atsma DE. Sustained effect of autologous bone marrow mono- nuclear cell injection in patients with refractory angina pectoris and chronic myocardial ischemia: twelve-month follow-up results. Am Heart J. 2006;
152:684-686.
3.
Murry CE, Field LJ, Menasche P. Cell- based cardiac repair: reflections at the 10-year point. Circulation. 2005;
112:3174-3183.
4.
Silva GV, Litovsky S, Assad JA, Sousa AL, Martin BJ, Vela D, Coulter SC, Lin J, Ober J, Vaughn WK, Branco RV, Oliveira EM, He R, Geng YJ, Willerson JT, Perin EC.
Mesenchymal stem cells differentiate into an endothelial phenotype, enhance vascular density, and improve heart function in a canine chronic ischemia model. Circulation. 2005; 111:150-156.
5.
Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, Martin BJ. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg.
2002; 73:1919-1925.
6.
Dai W, Hale SL, Martin BJ, Kuang JQ, Dow JS, Wold LE, Kloner RA. Allogeneic mesenchymal stem cell transplantation in postinfarcted rat myocardium: short- and long-term effects. Circulation.
2005; 112:214-223.
7.
Zhang S, Ge J, Sun A, Xu D, Qian J, Lin J, Zhao Y, Hu H, Li Y, Wang K, Zou Y. Com- parison of various kinds of bone mar- row stem cells for the repair of infarcted myocardium: Single clonally purified non-hematopoietic mesenchymal stem cells serve as a superior source. J Cell Biochem. 2006.
TJ, Discher DE, Sweeney HL. Mesenchy- mal stem cell injection after myocardial infarction improves myocardial compli- ance. Am J Physiol Heart Circ Physiol.
2006; 290:H2196-H2203.
9.
Chen SL, Fang WW, Ye F, Liu YH, Qian J, Shan SJ, Zhang JJ, Chunhua RZ, Liao LM, Lin S, Sun JP. Effect on left ventricular function of intracoronary transplan- tation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. Am J Cardiol. 2004; 94:92-95.
10.
Heeschen C, Lehmann R, Honold J, Assmus B, Aicher A, Walter DH, Martin H, Zeiher AM, Dimmeler S. Profoundly reduced neovascularization capacity of bone marrow mononuclear cells derived from patients with chronic ischemic heart disease. Circulation. 2004;
109:1615-1622.
11.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, Dim- meler S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res.
2001; 89:E1-E7.
12.
Meyerrose TE, Herrbrich P, Hess DA, Nolta JA. Immune-deficient mouse models for analysis of human stem cells. Biotechniques. 2003; 35:1262-1272.
13.
Yang Z, Berr SS, Gilson WD, Toufek- tsian MC, French BA. Simultaneous evaluation of infarct size and cardiac function in intact mice by contrast- enhanced cardiac magnetic resonance imaging reveals contractile dysfunction in noninfarcted regions early after myo- cardial infarction. Circulation. 2004;
109:1161-1167.
14.
Beeres SL, Atsma DE, van der LA, Pijnap- pels DA, van Tuyn J, Fibbe WE, de Vries AA, Ypey DL, van der Wall EE, Schalij MJ.
Human adult bone marrow mesen- chymal stem cells repair experimental conduction block in rat cardiomyo- cyte cultures. J Am Coll Cardiol. 2005;
46:1943-1952.
15.
smooth muscle-specific genes in pri- mary human cells after forced expres- sion of human myocardin. Cardiovasc Res. 2005.
16.
Tang YL, Zhao Q, Qin X, Shen L, Cheng L, Ge J, Phillips MI. Paracrine action enhances the effects of autologous mesenchymal stem cell transplantation on vascular regeneration in rat model of myocardial infarction. Ann Thorac Surg.
2005; 80:229-236.
17.
Fuster V, Sanz J, Viles-Gonzalez JF, Rajagopalan S. The utility of magnetic resonance imaging in cardiac tissue regeneration trials. Nat Clin Pract Car- diovasc Med. 2006; 3 Suppl 1:S2-S7.
18.
Anker SD, Sharma R. The syndrome of cardiac cachexia. Int J Cardiol. 2002;
85:51-66.
19.
Tang J, Xie Q, Pan G, Wang J, Wang M.
Mesenchymal stem cells participate in angiogenesis and improve heart function in rat model of myocardial ischemia with reperfusion. Eur J Cardio- thorac Surg. 2006; 30:353-361.
20.
Cai D, Marty-Roix R, Hsu HP, Spector M.
Lapine and canine bone marrow stromal cells contain smooth muscle actin and contract a collagen-glycosaminoglycan matrix. Tissue Eng. 2001; 7:829-841.
21.
Miano JM, Cserjesi P, Ligon KL, Pe- riasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res. 1994;
75:803-812.
22.
Frangogiannis NG, Michael LH, Entman ML. Myofibroblasts in reperfused myo- cardial infarcts express the embryonic form of smooth muscle myosin heavy chain (SMemb). Cardiovasc Res. 2000;
48:89-100.
23.
Mangi AA, Noiseux N, Kong DL, He HM, Rezvani M, Ingwall JS, Dzau VJ.
Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nature
24.
Nakamura Y, Wang XH, Xu CS, Asakura A, Yoshiyama M, From AHL, Zhang JY.
Xenotransplantation of long-term- cultured swine bone marrow-derived mesenchymal stem cells. Stem Cells.
2007; 25:612-620.
25.
Zhang S, Jia Z, Ge J, Gong L, Ma Y, Li T, Guo J, Chen P, Hu Q, Zhang P, Liu Y, Li Z, Ma K, Li L, Zhou C. Purified human bone marrow multipotent mesenchy- mal stem cells regenerate infarcted myocardium in experimental rats. Cell Transplant. 2005; 14:787-798.
26.
Vanoverschelde JL, Depre C, Gerber BL, Borgers M, Wijns W, Robert A, Dion R, Melin JA. Time course of functional recovery after coronary artery bypass graft surgery in patients with chronic left ventricular ischemic dysfunction.
Am J Cardiol. 2000; 85:1432-1439.
27.
Li JK. Comparative cardiac mechan- ics: Laplace’s Law. J Theor Biol. 1986;
118:339-343.
28.
Smaaland R, Laerum OD, Sothern RB, Sletvold O, Bjerknes R, Lote K. Colony- Forming Unit-Granulocyte-Macrophage and Dna-Synthesis of Human Bone- Marrow Are Circadian Stage-Dependent and Show Covariation. Blood. 1992;
79:2281-2287.
